Approaches to the control of human monocytic ehrlichiosis (HME), the most prevalent zoonotic ehrlichial disease reported in the USA to date, rely on better understanding of interactions between Ehrlichia chaffeensis, the etiologic agent, and its tick host. It has been demonstrated that several ixodid genera, including Amblyomma, Dermacentor and Ixodes, can be naturally infected with E. chaffeensis. Although detection of E. chaffeensis in naturally exposed ticks indicates susceptibility of these ticks to infection, it does not demonstrate their ability to transmit this pathogen to a susceptible vertebrate host. Repeated experimental transmission of an arthropod-borne pathogen by the suspected vector(s) is chief among the criteria that must be fulfilled before the competent vector(s) is (are) incriminated. The objective of this particular application, which is an essential step in determining the mechanisms behind tick transmission of E. chaffeensis, is to identify tick species and pathogen-acquisition scenarios that lead to effective experimental transmission of this pathogen. The central hypothesis of this project is that intrastadially infected adult male ixodid ticks are more effective experimental vectors of E. chaffeensis than adult female or trans-stadially infected adult male ticks. This central hypothesis will be tested through fulfillment of specific aims focused on (1) development of a PCR assay for E. chaffeensis that is sensitive enough to detect the pathogen within individual experimentally infected ticks, (2) intrastadial and transstadial tick-transmission of E. chaffeensis and (3) transmission of E. chaffeensis to multiple hosts by adult male ticks. The rationale for the proposed research is that it will lead to better understanding of the potential importance of different tick species and acquisition scenarios in the transmission of E. chaffeensis under natural conditions. Furthermore, once effective transmission cycles are identified, an experimental transmission model will be available to enable us to investigate interactions between E. chaffeensis and its tick host(s). This work is innovative because it questions the dogma that this tick borne pathogen is only transmitted transstadially, but this paradigm will be tested objectively with both intrastadial and transstadial transmission scenarios. In addition to development of a transmission model, the competence of several potential vectors will be determined, which is important for determining whether dogs could be a source of human infection with E. chaffeensis and for accurate diagnosis and prevention of HME. Collectively, these outcomes are expected to have significant positive effects on human (and animal) health, because they will lead to understanding the mechanisms responsible for transmission of E. chaffeensis and perhaps other closely related pathogens among human and domestic hosts.